We have previously shown that secretion of granules from cultured RBL-2H3 mast cells are dependent primarily on rise in cytosolic calcium and activation of protein kinase C (PKC) and that the phosphorylations of myosin heavy chains (MHC) by PKC and myosin light chains (MLC) by PKC and Ca2+-dependent myosin light chain kinase are temporally correlated with degranulation of RBL-2H3 cells when these cells are stimulated with antigen and other secretagogues. Also, the effects of selective blockade or induction of these phosphorylations by chemical agents supported the notion that degranulation requires phosphorylation of myosin by both PKC and MLCK as a potential mechanism for movement of granules to the cell surface. We have now extended our studies to include other possible regulators of vesicle trafficking with the ultimate aim of elucidating mechanisms for regulated secretion of preformed granules (i.e degranulation) and of newly generated Golgi-derived vesicles that contain cytokines (see report ZO1 HL 00990-10 LMI). By Western blot and RT-PCR we find that RBL-2H3 cells express at least one isoform of the proteins that comprise the SNARE complex, including N-ehtylmaleimide sensitive factor (NSF), Vesicle Associated Membrane Proteins (VAMP 1,2), cellubrevin, syntaxins 3 and 4, synaptotagmins 4 and 8 and SNAP proteins. A role for the SNARE complex is suggested by the following results. N-ethylmaleimide inhibits degranulation when induced by antigen (via the IgE recepetor) or by the receptor-independent stimulant thapsigargin. All detectable NSF is exclusively associated with the membrane fraction in RBL-2H3 cells and partially dissociates from the membrane fraction upon stimulation of the cells; in permeabilized cells and isolated membranes the protein could be released into the cytosol by Mg2+-ATP and high salt. Plasma membranes that were isolated by antibodies against either granulocyte membrane antigens or the surface IgE recepetor, attached to magnetic beads, also contain a dissociable NSF and the syntaxins. Co-distribution of the syntaxins and NSF with plasma membrane markers is also indicated by density gradient centrifugation. Therefore, RBL-2H3 cells contain all components of the SNARE complex that have been previously shown to regulate late events in calcium-dependent exocytosis in neuroendocrine cells.